T cell inducing vaccine containing an interepitope sequence that promotes antigen presentation

ABSTRACT

A vaccine contains a long-chain peptide antigen having a plurality of epitopes. An interepitope sequence located between two of the plurality of epitopes contains two to ten consecutive tyrosines, two to ten consecutive threonines, two to ten consecutive alanines, two to ten consecutive histidines, two to ten consecutive glutamines or two to ten consecutive asparagines. The vaccine may be an anticancer vaccine, an antibacterial vaccine or an antiviral vaccine. The vaccine may be a peptide vaccine, a DNA vaccine, an mRNA vaccine or a dendritic cell vaccine.

TECHNICAL FIELD

The present invention relates to a T cell inducing vaccine containing aninterepitope sequence that promotes antigen presentation.

BACKGROUND ART

The importance of cell-mediated immunity in tumor rejection by a cancerhost has been revealed as a result of long years of research related toimmune responses against cancer. In particular, it has been revealedthat CD8⁺ killer T cells (CD8⁺ cytotoxic T cells) are effector cellshaving an action of directly destroying tumors, that CD4⁺ helper T cellsare important regulatory cells that enhance the functions of CD8⁺ killerT cells and antigen-presenting cells, and that professionalantigen-presenting cells, such as dendritic cells and macrophages,stimulate T cells by presenting antigens thereto and activate T cellsvia costimulatory molecules, such as CD80, CD86, and cytokines, etc.,and the roles and positioning of the respective cells responsible forcellular immune responses against tumors have been established asdescribed below (Non-Patent Document 1).

Tumor cell derived proteins, after being phagocytosed byantigen-presenting cells, are cleaved into peptides of various lengthsby proteasomes, proteases, and peptidases within the cells. Among theresulting peptides, peptides of 8-10 amino acids are loaded as antigenepitope peptides onto major histocompatibility complex (MHC) class Imolecules and can be presented on the surfaces of the antigen-presentingcells. CD8⁺ killer T cells use T cell receptors (TCRs) to specificallyrecognize the MHC class I/antigenic peptide complexes and becomeactivated. The activated CD8⁺ killer T cells detect MHC classI/antigenic peptide complexes that are also present on tumor cells anddestroy the tumor cells using effector molecules, such as granzymes andperforin.

The function of CD4⁺ helper T cells is important for sufficientactivation of CD8⁺ killer T cells (Non-Patent Document 2). Antigenicproteins taken up by the antigen-presenting cells are cleaved intovarious lengths by proteases and peptidases within the cells and amongthe resulting antigenic peptides, those of 15-20 amino acids formcomplexes with MHC class II molecules and can be presented on theantigen-presenting cells. CD4⁺ helper T cells recognize thesespecifically and are activated. The activated CD4⁺ helper T cellsenhance differentiation, growth, and functions of CD8⁺ killer T cellsvia secretion of cytokines, such as interferon (IFN)-γ and interleukin(IL)-2. The CD4⁺ helper T cells also have a function of activatingantigen-presenting cells via a CD40 ligand/CD40 pathway, and theantigen-presenting cells activated by the CD4⁺ helper T cells areimproved in the capability to stimulate CD8⁺ killer T cells (Non-PatentDocument 3). It is well known from before that CD4⁺ helper T cells alsohave an action of enhancing antigen-specific IgG antibody production inB cells.

Based on the above understanding of T-cell immune response, a cancervaccine therapy has been conceived where a tumor specific antigen isrepeatedly administered as a vaccine antigen to induce tumor-specificCD8⁺ killer T cells within a patient's body to suppress the growth,metastasis, and recurrence of cancer. Various forms of the antigen ofthe cancer vaccines are known, such as synthetic peptides, recombinantproteins, processed cells. The present inventors have previouslyprepared a cancer vaccine using a full-length recombinant protein of atumor antigenic protein as the antigen. The full-length protein includesdiverse antigenic peptides recognized by CD8⁺ killer T cells and CD4⁺helper T cells and is expected to activate both types of T cells at thesame time. However, with an exogenous (extracellular) antigenic protein,although the activation of CD4⁺ helper T cells via the MHC class IIpathway proceeds readily, the activation of CD8⁺ killer T cells via theMHC class I pathway does not proceed readily. This is due to reasons ofmechanisms of uptake and antigen processing of exogenous antigenicproteins in antigen-presenting cells (Non-Patent Document 4).

Therefore many attempts are being made in and outside Japan tochemically synthesize short chain peptides, mainly, epitope peptides of8 to 10 residues recognized by CD8⁺ killer T cells and clinically applyvaccines using these peptides as antigens. With short peptide antigens,presentation to T cells occurs readily because such peptides binddirectly to MHC molecules on cell surfaces without undergoing uptake andantigen processing within antigen-presenting cells. Also, short chainpeptides can be manufactured by chemical synthesis and has the advantageof being simpler to manufacture than recombinant proteins, whichrequires the use of genetically modified organisms.

However, immunological problems have been pointed out in regard to thedirect binding of short peptide antigens to MHC molecules on cellsurfaces without undergoing uptake and antigen processing withinantigen-presenting cells (Non-Patent Document 5). Exogenous antigenicproteins are phagocytosed by professional antigen-presenting cells, suchas dendritic cells and macrophages, that are provided with costimulatorymolecules (CD80, CD86, etc.) and are processed within the cells, andantigen presentation to T cells is performed in a mode with appropriateconcentration and costimulation. On the other hand, short peptideantigens bind directly to MHC molecules on cell surfaces and thereforeeven general somatic cells, which do not have uptake ability (phagocyticability) and do not express costimulatory molecules, can present theshort peptide antigens in a massive, inappropriate mode that lackscostimulation. In this case, the T cells that recognize the complexes ofthe short peptide antigens and MHCs become prone to depletion andapoptosis and this can consequently lead to immunological tolerance tothe targeted antigen.

In view of such problems of short chain peptide vaccines, the usefulnessof long chain synthetic peptide antigens is attracting attention(Non-Patent Document 5). Along chain peptide antigen is a polypeptidehaving several dozen residues such that include two or more T cellrecognition epitope peptides. Unlike a short chain peptide, a long chainpeptide antigen cannot bind directly in intact form to an MHC molecule.As with protein antigens, long chain peptide antigens undergo uptake andintracellular processing by professional antigen-presenting cells withphagocytic ability, such as dendritic cells and macrophages, and the Tcell epitope peptides included in the long chain peptide antigens formcomplexes with MHC molecules only thereafter and are thus presented to Tcells in a mode with appropriate concentration and costimulation. Longchain peptide antigens do not function as vaccine antigens with generalsomatic cells lacking antigen phagocytic ability and therefore, unlikeshort chain peptide vaccines, do not give rise to inappropriate antigenpresentation to T cells. Moreover, chemical synthetic methods can beused to manufacture long chain peptide antigens and therefore, as withshort peptide antigens, the advantage of being comparatively easy tomanufacture is also provided.

Long chain peptide antigens manufactured by chemical synthesis also havea major advantage in that it is possible to freely design the sequence.A long chain peptide antigen is designed so that two or more T cellepitopes are included within a single peptide, and these T cell epitopesmay be derived from a single cancer antigenic protein or may be derivedfrom a plurality of cancer antigenic proteins. Also, the T cell epitopesmay be restrictive to a single MHC or may be restrictive to a pluralityof MHCs. It is also possible to design so that a long chain peptideantigen includes an epitope recognized by a CD8⁺ killer T cell and anepitope recognized by a CD4⁺ helper T cell at the same time. Long chainpeptide antigens can thus serve as high performance vaccine antigensthat can induce diverse T cells. However, for the set of epitopescontained in a long chain peptide antigen to be presented to T cellsefficiently, the epitopes must be cut out as epitope peptides of lengthsand sequences enabling binding with MHC molecules by sequences betweenthe respective epitopes on the long chain peptide antigen being cleavedappropriately by proteasomes, proteases, and peptidases in anantigen-presenting cell based on the mechanism of antigen presentationreactions.

In regard to MHC class II binding epitope peptides recognized by CD4⁺helper T cells, the terminuses of the epitope peptide binding groove onan MHC class II molecule are in an open state and epitope peptides ofvarious lengths can bind to the MHC class II molecule (Non-PatentDocument 6). Therefore, with MHC class II binding epitope peptides, therestriction of length is comparatively relaxed. On the other hand, inregard to MHC class I binding epitope peptides recognized by CD8⁺ killerT cells, the terminuses of the epitope peptide binding groove on an MHCclass I molecule are in a closed state and only epitope peptides,strictly restricted to 8 to 10 residues, can bind to the MHC class Imolecule. It is thus especially important with MHC class I bindingepitope peptides that peptides of appropriate lengths are produced inantigen-presenting cells.

The lengths and sequences of the epitope peptides that bind to MHCmolecules are determined by complex cleavage reactions involvingintracellular proteasomes and various proteases and peptidases. In theproduction of MHC class I binding epitope peptides, proteasomes presentin the cytoplasm first perform rough cleavage of the antigenic proteinor long chain peptide antigen. The terminuses of the resulting peptidefragments are cleaved by other proteases and peptidases based on certainsubstrate sequence specificities and trimmed to appropriate lengths(trimming reactions). Although a group of enzymes that trim theN-terminuses of the peptide fragments in this process exists, enzymesthat trim the C-terminuses are unknown, and determination of theC-terminuses of the MHC class I binding epitope peptides is dependentonly on the initial cleavage reactions by the proteasomes (Non-PatentDocument 7). However, the substrate sequence specificities ofproteasomes have not been revealed in detail and it is difficult topredict peptide sequences that can be cleaved readily by proteasomes.

In view of the above epitope production mechanism, how the sequencesbetween the epitopes included in the long chain peptide antigen arecleaved by the intracellular proteasomes, proteases, and peptidasesstrongly influences the production of the preceding and subsequentepitope peptides and is consequently considered to be an extremelyimportant factor that defines the induction of T cells by vaccines usinglong chain peptide antigens.

PRIOR ART DOCUMENTS Non-Patent Documents

-   Non-Patent Document 1: Ribas, A., et al., Clin. Oncol. 2003; 21(12):    2415-2432-   Non-Patent Document 2: Shiku, H., Int. J. Hematol. 2003; 77(5):    435-8.-   Non-Patent Document 3: Behrens, G., et al., Immunol. Cell Biol.

2004; 82(1): 84-90

-   Non-Patent Document 4: Shen, L. & Rock, K. L., Curr. Opin. Immunol.    2006; 18(1): 85-91-   Non-Patent Document 5: Melief, C. J. M., & van der Burg, S. H.,    Nature Rev. Cancer, 2008; 8(5): 351-360.-   Non-Patent Document 6: Holland, C. J., et al., Front Immunol. 2013;    4: 172.-   Non-Patent Document 7: Goldberg, A. L., et al., Mol. Immunol. 2002;    39(3-4): 147-64.-   Non-Patent Document 8: Muraoka, D., et al., Vaccine. 2013; 31:    2110-2118.

SUMMARY OF THE INVENTION Problem to be Solved by the Invention

For the T cell epitope peptides included in a certain long chain peptideor protein to be efficiently presented as antigens, the epitope peptidesequences must be cut out appropriately from the long chain peptide orprotein by intracellular proteasomes, proteases, and peptidases. Forthis purpose, it is necessary for the sequences between the epitopes toaptly include recognition sites for the proteasomes, proteases, andpeptidases.

In conventional arts, it was hardly examined what sort of interepitopesequence would satisfy the above condition. Therefore, with a vaccineusing a long chain peptide antigen designed without examining theinterepitope sequence, the induction of T cells that recognize theincluded epitope peptides is weak or cannot be confirmed in some cases.

The present invention has been made in view of the circumstancesdescribed above, and an object thereof is to provide, in a long chainpeptide antigen containing a plurality of epitope peptides, aninterepitope sequence that effectively achieves antigen presentation ofthe respective epitope peptides.

Means for Solving the Problem

In a vaccine including a long chain peptide antigen having a pluralityof epitopes according to the present invention for achieving the aboveobject, each interepitope sequence is one selected from a groupconsisting of two to ten consecutive tyrosines, two to ten consecutivethreonines, two to ten consecutive alanines, two to ten consecutivehistidines, two to ten consecutive glutamines, and two to tenconsecutive asparagines and it is especially preferable for the sequenceto be tyrosines, glutamines, or asparagines. Here, the number ofconsecutive tyrosines, consecutive threonines, consecutive histidines,consecutive glutamines, or consecutive asparagines is preferably four toeight, more preferably four to six, and especially six.

By having the above arrangement, the long chain peptide antigen iscleaved inside a body by enzymes within a living body so that therespective epitopes can perform antigen presentation and the respectiveepitopes thus exhibit antigen presenting abilities effectively. Also,with a vaccine using along chain peptide antigen having an interepitopesequence constituted of consecutive tyrosines, uptake intoantigen-presenting cells is also improved.

The vaccine is preferably one selected from the group consisting ofanticancer vaccines (including dendritic cell vaccines), antibacterialvaccines, and antiviral vaccines.

Also, the vaccine is preferably at least one selected from the groupconsisting of peptide vaccines, DNA vaccines, mRNA vaccines, anddendritic cell vaccines. In a case of a dendritic cell vaccine, apeptide antigen or mRNA is added.

Also in a case where the vaccine is a peptide vaccine, it is preferablyarranged as a vaccine in combination with a hydrophobizedpolysaccharide, especially, cholesterol-modified pullulan (CHP) as adelivery system.

Effect(s) of the Invention

With the present invention, the plurality of epitopes within the vaccinecan perform antigen presentation effectively and therefore a vaccinehaving a high effect can be provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 The influences of differences in interepitope sequence of longchain peptide vaccines, containing a plurality of CD8⁺ T cell epitopes,on specific CD8⁺ T cell induction by the vaccines were examined. Longchain peptide antigens MEN, all containing three types of mouse CD8⁺ Tcell epitope sequences (MA p265, NY p81, and mERK2 9m), weresynthesized. The sequence between the respective epitopes was set to oneof six consecutive tyrosines (Y₆), glycines (G₆), prolines (P₆), orthreonines (T₆). Each long chain peptide antigen was complexed withcholesterol-modified pullulan (CHP), which is a type of delivery system,and administered as a vaccine to a mouse. In the process ofadministration, CpG oligo DNA was coadministered as an adjuvant. Spleencells were collected one week after the final administration and thefrequencies of CD8⁺ T cells specific to the respective epitope sequenceswere measured by an intracellular cytokine staining method.

FIG. 2 The influences of differences in interepitope sequence of longchain peptide vaccines, containing a plurality of CD8⁺ T cell epitopes,on therapeutic effects of the vaccines were examined. Long chain peptideantigens MEN, all containing three types of mouse CD8⁺ T cell epitopesequences (MA p265, NY p81, and mERK2 9m), were synthesized. Thesequence between the respective epitopes was set to one of sixconsecutive tyrosines (Y₆), glycines (G₆), or prolines (P₆). Each longchain peptide antigen was complexed with CHP and administered in asingle dose as a vaccine to a mouse. As a control, a short chain peptidevaccine, constituted of just the mERK2 9m peptide, was mixed withFreund's incomplete adjuvant and administered. In the process ofadministration, CpG oligo DNA was coadministered as an adjuvant. On theday after administration, a mouse fibrosarcoma cell line CMS5a,expressing mERK2 as a tumor antigen and presenting the CD8⁺ T cellepitope mERK2 9m derived from the same antigen, was implantedsubcutaneously and its growth was recorded over time.

FIG. 3 The influences of differences in interepitope sequence of longchain peptide vaccines, containing a plurality of CD8⁺ T cell epitopes,on specific CD8⁺ T cell induction by the vaccines were examined. Longchain peptide antigens NME, all containing three types of mouse CD8⁺ Tcell epitope sequences (MA p265, NY p81, and mERK2 9m), weresynthesized. The antigens differ from the MEN in FIG. 1 in the order ofthe three types of epitopes. The sequence between the respectiveepitopes was set to one of six consecutive tyrosines (Y₆), glycines(G₆), or prolines (P₆). Vaccines containing the respective long chainpeptide antigens were administered to mice in the same manner as in FIG.1 and the frequencies of CD8⁺ T cells specific to the respective epitopesequences were measured by the intracellular cytokine staining method.

FIG. 4 Whether or not the usefulness of an interepitope sequence,constituted of consecutive tyrosines, is influenced by preceding andsubsequent epitope sequences was examined. Long chain peptide antigensMEN, ENM, and NME, all containing three types of mouse CD8⁺ T cellepitope sequences (MA p265, NY p81, and mERK2 9m), were synthesized.MEN, ENM, and MEN differ in the order of the three types of epitopes.The sequence between the respective epitopes was set to six consecutivetyrosines (Y₆). Vaccines containing the respective long chain peptideantigens were administered to mice in the same manner as in FIG. 1 andthe frequencies of CD8⁺ T cells specific to the respective epitopesequences were measured by the intracellular cytokine staining method.

FIG. 5 The influences of the difference between a native sequence and aconsecutive tyrosine sequence as the interepitope sequence on specificCD8⁺ T cell induction and specific CD4⁺ T cell induction by vaccineswere examined. A long chain peptide antigen ESO1 LP (native type) and along chain peptide antigen ESO1 LP (Y₆), both containing a mouse CD8⁺ Tcell epitope sequence (NY p81) and a mouse CD4⁺ T cell epitope sequence(NY p91) that are derived from human NY-ESO-1 antigen, were synthesized.As the sequence between epitopes, the native amino acid sequence ofNY-ESO-1 was retained with ESO1 LP (native type) and the sequence of sixconsecutive tyrosines (Y₆) was used with ESO1 LP (Y₆). Vaccinescontaining the respective long chain peptide antigens were administeredto mice in the same manner as in FIG. 1 and the frequencies of CD8⁺ Tcells and CD4⁺ T cells specific to the respective epitope sequences weremeasured by the intracellular cytokine staining method.

FIG. 6 For interepitope sequences constituted of consecutive tyrosines,the relationship between the number of tyrosines and specific T cellinduction by vaccines were examined. Long chain peptide antigens MEN,all containing three types of mouse CD8⁺ T cell epitope sequences (MAp265, NY p81, and mERK2 9m), were synthesized. The sequence between therespective epitopes was set to one to six consecutive tyrosines.Vaccines containing the respective long chain peptide antigens wereadministered to mice in the same manner as in FIG. 1 and the frequenciesof CD8⁺ T cells specific to the respective epitope sequences weremeasured by the intracellular cytokine staining method.

FIG. 7 For interepitope sequences constituted of consecutive tyrosines,the relationship between the number of tyrosines and specific T cellinduction by vaccines were examined. Long chain peptide antigens MEN,all containing three types of mouse CD8⁺ T cell epitope sequences (MAp265, NY p81, and mERK2 9m), were synthesized. The sequence between therespective epitopes was set to five to ten consecutive tyrosines.Vaccines containing the respective long chain peptide antigens wereadministered to mice in the same manner as in FIG. 1 and the frequenciesof CD8⁺ T cells specific to the respective epitope sequences weremeasured by the intracellular cytokine staining method.

FIG. 8 The influences of differences in interepitope sequence of longchain peptide vaccines on uptake of the vaccines into antigen-presentingcells were examined. Long chain peptide antigens MEN, all containingthree types of mouse CD8⁺ T cell epitope sequences (MA p265, NY p81, andmERK2 9m), were synthesized. The sequence between the respectiveepitopes was set to one of six consecutive tyrosines (Y₆), glycines(G₆), or prolines (P₆). Each long chain peptide antigen, labeled withthe fluorescent dye FAM, was complexed with CHP and administered invitro to mouse dendritic cells and mouse macrophages. After 60 minutes,the fluorescence uptakes into the respective cells were measured by flowcytometry with the P5 fraction in the figure being deemed to correspondto the dendritic cells and the P₆ fraction in the figure being deemed tocorrespond to the macrophages.

FIG. 9 The influences of differences in interepitope sequence of longchain peptide vaccines on uptake of the vaccines into antigen-presentingcells were examined. The same FAM-labeled long chain peptide antigens asthose in FIG. 8 were complexed with CHP and administered subcutaneouslyto mice. After 16 hours, cells were collected from a regional lymph nodeof the administration site, and the fluorescence uptakes into dendriticcells and mouse macrophages were measured by flow cytometry with the P4fraction in the figure being deemed to correspond to the dendritic cellsand the P5 fraction in the figure being deemed to correspond to themacrophages.

FIG. 10 The influences of differences in interepitope sequence of longchain peptide vaccines, containing a plurality of CD8⁺ T cell epitopes,on specific CD8⁺ T cell induction by the vaccines were examined. Longchain peptide antigens NMW, all containing three types of human CD8⁺ Tcell epitope sequences (NY p157:HLA-A0201 restrictive, MA4p143:HLA-A2402 restrictive, and WT1: HLA-A2402 restrictive p235), weresynthesized. The sequences between the respective epitopes were set tothose of six consecutive amino acids shown in the figure. Each longchain peptide antigen was complexed with cholesterol-modified pullulan(CHP), which is a type of delivery system, and administered in vitro asa vaccine to an immortalized human B cell line (LCL). Using this as theantigen-presenting cells, co-culturing with CD8⁺ T cell clone 1G4 cellsspecific to NY p157 was performed and the activation of the 1G4 cellsdue to antigen presentation was measured by an IFN-γ ELISPOT method. Asa positive control, LCL administered with an NY p157 short chain peptidewas used as antigen-presenting cells, and as a negative control, LCLwithout antigen added was used as antigen-presenting cells.

FIG. 11 The influences of differences in interepitope sequence of RNAvaccines, containing a plurality of CD8⁺ T cell epitopes, on specificCD8⁺ T cell induction by the vaccines were examined. mRNAs encoding longchain peptide antigens NMW, all containing three types of human CD8⁺ Tcell epitope sequences (NY p157:HLA-A0201 restrictive, MA4p143:HLA-A2402restrictive, and WT1: HLA-A2402 restrictive p235), were synthesized. Thesequences between the respective epitopes were set to those of sixconsecutive amino acids shown in the figure. Each mRNA was introduced invitro as a vaccine into LCL by an electroporation method. Using this asthe antigen-presenting cells, co-culturing with CD8⁺ T cell clone 1G4cells specific to NY p157 or CD8⁺ T cell clone RNT007#45 cells specificto M4A p143 was performed and the activation of the CD8⁺ T cells due toantigen presentation was measured by the IFN-γ ELISPOT method.

MODES FOR CARRYING OUT THE INVENTION

Although embodiments of the present invention shall be described withreference to the drawings, the technical scope of the present inventionis not restricted to these embodiments and the invention may be carriedout in various modes without changing the gist of the invention. Also,the technical scope of the present invention extends to the range ofequivalents.

<Materials and Methods>

(1) Test Animals

Six- to twelve-week-old female BALB/c mice were purchased from JapanSLC, Inc. and reared at the Animal Center of Mie University Faculty ofMedicine. The animal experiment protocol was approved by the EthicsCommittee of Mie University Faculty of Medicine.

(2) Peptides

Synthetic long chain peptides were purchased from Bio-Synthesis Inc. Thesequences of the synthetic long chain peptides were as follows.

MEN(Y₆): (Sequence No. 1) SNPARYEFLYYYYYYQYIHSANVLYYYYYYRGPESRLLMEN(G₆): (Sequence No. 2) SNPARYEFLGGGGGGQYIHSANVLGGGGGGRGPESRLLMEN(P₆): (Sequence No. 3) SNPARYEFLPPPPPPQYIHSANVLPPPPPPRGPESRLLMEN(T₆): (Sequence No. 4) SNPARYEFLTTTTTTQYIHSANVLTTTTTTRGPESRLLNME(Y₆): (Sequence No. 5) RGPESRLLYYYYYYSNPARYEFLYYYYYYQYIHSANVLNME(G₆): (Sequence No. 6) RGPESRLLGGGGGGSNPARYEFLGGGGGGQYIHSANVLNME(P₆): (Sequence No. 7) RGPESRLLPPPPPPSNPARYEFLPPPPPPQYIHSANVLENM(Y₆): (Sequence No. 8) QYIHSANVLYYYYYYRGPESRLLYYYYYYSNPARYEFLMEN(Y₁):  (Sequence No. 9) SNPARYEFLYQYIHSANVLYRGPESRLL MEN(Y₂): (Sequence No. 10) SNPARYEFLYYQYIHSANVLYYRGPESRLL MEN(Y₃): (Sequence No. 11) SNPARYEFLYYYQYIHSANVLYYYRGPESRLL MEN(Y₄):(Sequence No. 12) SNPARYEFLYYYYQYIHSANVLYYYYRGPESRLL MEN(Y₅):(Sequence No. 13) SNPARYEFLYYYYYQYIHSANVLYYYYYRGPESRLL MEN(Y₈):(Sequence No. 14) SNPARYEFLYYYYYYYYQYIHSANVLYYYYYYYYRGPESRLL MEN (Y₁₀):(Sequence No. 15) SNPARYEFLYYYYYYYYYYQYIHSANVLYYYYYYYYYYRGPESRLLESO1 LP (native type): (Sequence No. 16)GARGPESRLLEFYLAMPFATPMEAELARRSLAQDAPPLPV ESO1 LP (Y₆): (Sequence No. 17)GPESRLLYYYYYYYLAMPFATPMEAELARRSLA NMW(A₆): (Sequence No. 18)SLLMWITQCAAAAAANYKRCFPVIAAAAAACMTWNQMNL NMW(E₆): (Sequence No. 19)SLLMWITQCEEEEEENYKRCFPVIEEEEEECMTWNQMNL NMW(G₆): (Sequence No. 20)SLLMWITQCGGGGGGNYKRCFPVIGGGGGGCMTWNQMNL NMW(H₆): (Sequence No. 21)SLLMWITQCHHHHHHNYKRCFPVIHHHHHHCMTWNQMNL NMW(N₆): (Sequence No. 22)SLIWITQCNNNNNNNYKRCFPVINNNNNNCMTWNQMNL NMW(P₆): (Sequence No. 23)SLLMWITQCPPPPPPNYKRCFPVIPPPPPPCMTWNQMNL NMW(Q₆): (Sequence No. 24)SLLMWITQCQQQQQQNYKRCFPVIQQQQQQCMTWNQMNL NMW(S₆): (Sequence No. 25)SLLMWITQCSSSSSSNYKRCFPVISSSSSSCMTWNQMNL NMW(Y₆): (Sequence No. 26)SLLMWITQCYYYYYYNYKRCFPVIYYYYYYCMTWNQMNL

Synthetic short chain peptides were purchased from Sigma Genosys. Theamino acid sequences of the peptides were as follows.

(Sequence No. 27) MA p265: SNPARYEFL (Sequence No. 28) mERK2 9m:QYIHSANVL (Sequence No. 29) NY p81: RGPESRLL (Sequence No. 30) NY p157:SLLMWITQC

Template cDNAs used to synthesize the RNA vaccines were purchased fromOperon Biotechnologies, Inc. The sequences of the cDNAs were as follows.

NMW (Y₆): (Sequence No. 31)GGATCCATGAGCCTCCTGATGTGGATTACCCAATGCTATTACTACTATTACTACAACTATAAGAGATGTTT CCCCGTGATCTATTACTACTACTACTATTGCTATACATGGAATCAGATGAACCTGTGAGAATTC NMW (T₆): (Sequence No. 32)GGATCCATGAGCCTGCTCATGTGGATCACACAATGCACCACTACTACCACAACCAACTACAAGAGATGT TTCCCCGTGATTACCACAACCACAACTACGTGCTATACGTGGAATCAGATGAACCTGTGAGAATTC NMW (G₆): (Sequence No. 33)GGATCCATGAGCTTGCTCATGTGGATCACCCAATGTGGAGGAGGTGGTGGAGGCAACTACAAGCGATGTTTCCCCGTGATAGGCGGTGGAGGTGGAGGGTGCTACACATGGACCAGATGACCTGTGAGATTC NMW (P₆): (Sequence No. 34)GGATCCATGAGTCTGCTGATGTGGATCACTCAGTGTCCTCCACCACCACCACCCAACTACAAGAGGTGT TTCCCCGTGATTCCACCACCTCCTCCTCCATGCTATACCTGGAATCAGATGAACCTGTGAGAATTC

(3) Other Reagents

Cholesterol-modified pullulan (abbreviation CHP) (CHP-80T) was obtainedfrom NOF Corporation. CpG oligo DNA was purchased from Hokkaido SystemScience Co., Ltd. FITC-labeled anti-CD4 monoclonal antibody (cloneRM4-5), PerCP-Cy5.5-labeled anti-CD8 monoclonal antibody (clone 53-6.7),and APC-labeled anti-IFN-γ antibody (clone XMG1.2) were purchased fromeBiosciece Inc. or BD Biosciences. Anti-human IFN-γ antibody andbiotinylated anti-human IFN-γ antibody was purchased from Mabtech AB.

(4) Preparation of Complexes of Long Chain Peptide Antigens and CHP

Each long chain peptide was dissolved in dimethyl sulfoxide (DMSO) at aconcentration of 10 mg/mL. CHP was dissolved in 6M urea-containingphosphate buffered saline (PBS) at a concentration of 10 mg/mL. 1 mL (10mg) of the long chain peptide solution and 20 mL (200 mg) of the CHPsolution were mixed and left to stand at room temperature overnight in adark place. The liquid mixture was transferred into a dialysis membrane(molecular weight cutoff: 3,500; Thermo Fisher Scientific, Inc.) anddialyzed for 2 hours to overnight at 4° C. against 0.6M urea-containingPBS of a volume ratio of not less than 100 times as the dialysis outersolution. Dialysis was then performed for 2 hours to overnight at 4° C.against 0.06M urea-containing PBS of a volume ratio of not less than 100times as the dialysis outer solution. Dialysis was performed again for 2hours to overnight at 4° C. against PBS of a volume ratio of not lessthan 100 times as the dialysis outer solution. The dialyzed innersolution was collected, filtered through a filtration sterilizationfilter of 0.45 μm or 0.22 μm pore size, and thereafter the UV absorptionat 280 nm was measured to determine the final concentration of the longchain peptide from its molecular extinction coefficient.

(5) Administration of Vaccines to Mice and Separation of Spleen Cells

Each CHP/long chain peptide complex as the vaccine and the CpG oligo DNAas the adjuvant were administered at the same time to a mouse.Administration was performed by subcutaneous injection on the back ofthe mouse. As the dose, the CHP/long chain peptide complex wasadministered at 0.05 to 0.1 mg equivalent of long chain peptide peradministration. The CpG oligo DNA was administered at 0.05 mg peradministration. One week after the final administration, spleen cellswere separated by the following procedure from each vaccine-administeredmouse. The spleen was isolated from the mouse and removed of blood byrinsing with RPMI1640 medium. After the spleen was triturated using aglass slide, the released cells were collected in RPMI1640 medium. Aftercentrifuging (400×g, 5 minutes, 4° C.), the supernatant was removed andthe cells were treated for 1 minute by adding 2 mL of ACK solution. 18mL of RPMI1640 medium were added and centrifugation (400×g, 5 minutes,4° C.) was performed. The supernatant was removed and the cells weresuspended in RPMI1640 medium of an appropriate amount. After countingthe number of cells, the cells were suspended in RPMI1640 mediumcontaining 10% fetal bovine serum (FBS) so that the cell concentrationwas 1×10⁷ cells/mL.

(6) Intracellular Cytokine Staining of Mouse Spleen Cells

The mouse spleen cells were added at 5×10⁶ cells/0.5 mL per well to a24-well culture plate (Nunc). NY p81, MAGE p265, or mERK2 9m as theshort chain peptide for CD8⁺ T cell stimulation or ESO1 LP (native type)or ESO1 LP (Y₆) CD4⁺ T cell stimulation was added at a concentration of10 μM and culturing under 37° C. and 5% CO₂ was performed for 6 hours.Thereafter, GoldiPlug (BD Biosciences), diluted 10-fold with 10%FBS-containing RPMI1640 medium, was added at 50 μL per well andculturing under 37° C. and 5% CO₂ was performed for 6 hours. The cellswere collected and transferred to a 96-well round bottom microplate(Nunc). After centrifuging (1200 rpm, 1 minute, 4° C.) and removing thesupernatant, the cells were suspended in 50 μL of staining buffer (PBScontaining 0.5% bovine serum albumin) per well. The FITC-labeledanti-CD8 antibody or the FITC-labeled anti-CD4 antibody was added andafter mixing, the cells were left to stand for 15 minutes in a darkplace at 4° C. After rinsing the cells twice with 200 μL of the stainingbuffer, 100 μL of Cytofix/Cytoperm buffer (BD Biosciences) were addedand mixed gently. After leaving to stand for 20 minutes in a dark placeat room temperature, rinsing with 100 μL of Perm/Wash buffer (BDBiosciences) was performed twice. 50 μL of Perm/Wash buffer with therespective types of anti-cytokine antibodies added were added to thecells and after suspending gently, the cells were left to stand for 15minutes in a dark place at room temperature. After rinsing twice with100 μL of Perm/Wash buffer, the cells were re-suspended in 200 μL of thestaining buffer and transferred to a round-bottom polystyrene tube (BDBiosciences). The cells were analyzed by a flow cytometer (FACS CantoII, BD Biosciences) using the included analysis software (FACSDiva).

(7) Mouse Tumor Growth Test

A subcloned CMS5a cell line, obtained from a CMS5 cell line isolatedfrom fibrosarcoma induced by administering 3-methylcholanthrene to aBALB/c mouse, expresses mutant ERK2 (mERK2) as a tumor antigen andpresents a CD8⁺ T cell epitope derived from the mERK2. The CMS5a cellline cultured in a T75 flask (Nunc) was detached using PBS containing0.5% trypsin and collected in RPMI1640 medium containing 10% FBS. Aftercentrifuging (400×g, 5 minutes, 4° C.), the supernatant was removed, andthe cells were rinsed twice with RPMI1640 medium, thereafter suspendedin RPMI1640 medium at a concentration of 1×10⁶ cells/100 μL, andimplanted subcutaneously in BALB/c mice at a dose of 100 μL/individual.The CHP/long chain peptide complexes and the adjuvant were administered7 days before tumor implantation (prophylactic condition). After tumorimplantation, the length and breadth of the tumor were measured and theproduct thereof was recorded as tumor size. The data in the tumor growthtest were compared by Student's t test using Microsoft Excel (MicrosoftCorporation).

(8) Uptake of Long Chain Peptide Antigens by Antigen Presenting Cells

In vitro uptake of the long chain peptide antigens by antigen-presentingcells was measured as follows. Each long chain peptide labeled with afluorescent dye was complexed with CHP by the method described above.Spleen cells separated from a normal mouse were added at 1×10⁶ cells/0.5mL/well to a 24-well plate. Each CHP/fluorescent-labeled long chainpeptide complex was added at a concentration of 10 μg/mL and culturingwas performed at 37° C. Cells were collected after 60 minutes andstained with an anti-CD11c antibody and an anti-F4/80 antibody. Theuptake of the fluorescence-labeled long chain peptide antigens by CD11c⁺cells (dendritic cells) and F4/80⁺ cells (macrophages) were observedusing flow cytometry.

A test of uptake of long chain peptide antigens by antigen-presentingcells in individual animals was performed as follows. Each long chainpeptide was fluorescence-labeled, complexed with a CHP nanogel,administered subcutaneously to BALB/c mice. 16 hours afteradministration, cells were collected from lymph nodes and after stainingwith the anti-CD11c antibody and the anti-F4/80 antibody, the uptake ofthe fluorescence-labeled long chain peptide antigens by CD11c⁺ cells(dendritic cells) and F4/80⁺ cells (macrophages) was analyzed by flowcytometry.

(9) Administration of Long Chain Peptide Vaccines to Immortalized B CellLine (LCL)

Cryostored LCL was rinsed with RPMI medium and suspended at 1.25×10⁶/mLin X-VIVO15 medium. This was dispensed in 0.4 mL aliquots intopolypropylene tubes, and 0.1 mL of a vaccine solution (0.1 mg/mL aspeptide) was added to each tube. The cells were cultured for 24 hours at37° C. in the presence of 5% CO₂ and then used as antigen-presentingcells.

(10) Administration of RNA Vaccines to Immortalized B Cell Line (LCL)

mRNA was introduced by an electroporation method (300V, 700 μs) usingECM830 into LCL that was rinsed and suspended in the same manner as in(9). The cells were cultured for 24 hours at 37° C. in the presence of5% CO₂ and then used as antigen-presenting cells.

(11) ELISPOT Method

75 μL aliquots of anti-IFN-γ antibody for capture, diluted to anappropriate concentration, were dispensed into a 96-well plate(Millipore Corp., Multiscreen HA, MAHAS4510) specially designed forELISPOT and left to stand overnight at 4° C. After discarding the liquidand rinsing with RPMI medium, 100 μL aliquots of RPMI medium containing10% fetal bovine serum were dispensed and the plate was left to standfor not less than 1 hour at 37° C. The liquid was discarded, and the LCLprepared in (9) or (10) was adjusted to 5×10⁴ cells/100 μL/well andadded to each well. The cryostored CD8⁺ T cell clones were thawed,rinsed, adjusted to 5×10⁵/mL with RPMI medium, and thereafter added in0.1 mL aliquots to each well. After culturing for 24 hours at 37° C. inthe presence of 5% CO₂, the liquid was discarded and the plate wasrinsed well with phosphate buffered saline containing 0.05% Tween 20(PBS-T). A biotin-labeled IFN-γ antibody for detection was diluted to anappropriate concentration and dispensed in 0.1 mL aliquots into eachwell. After letting stand overnight at 4° C., the plate was rinsed wellwith PBS-T, and an alkaline phosphatase-labeled streptavidin diluted toan appropriate concentration was added in 0.1 mL aliquots. Afterincubating for 1 hour at room temperature, the plate was rinsed wellwith PBS-T. A coloring solution was added in 0.1 mL aliquots and allowedto react for 5 minutes to 30 minutes at room temperature. When theformation of spots was observed, the reaction was stopped by rinsingwith water.

(12) Preparation of mRNAs Encoding Long Chain Peptide Antigens

cDNAs encoding the intended long chain peptide antigens were purchasedas synthetic genes from Operon Biotechnologies, Inc. Each of these wascloned into the multiple cloning site of a pcDNA3.1 vector. The primingsite on the T7 promoter contained in the pcDNA3.1 was used to synthesizemRNA by a conventional method using MEGAscript (registered trademark) T7Transcription Kit, made by Life Technologies, Inc., etc.

<Test Results>

FIG. 1 shows that with vaccines having a long chain peptide, whichcontains a plurality of T cell epitopes, as an antigen, differences ininterepitope sequence influence the success or failure of specific Tcell induction by the respective epitopes. The long chain peptideantigens MEN, all containing the three types of mouse CD8⁺ T cellepitope sequences, MA p265, NY p81, and mERK2 9m, which are derived fromthe human tumor antigens MAGE-A4 and NY-ESO-1 and the mouse tumorantigen, mutant ERK2 (mERK2), were synthesized. The sequence between thethree types of epitopes was set to one of six consecutive tyrosines(Y₆), glycines (G₆), prolines (P₆), or threonines (T₆). Each long chainpeptide antigen was complexed with cholesterol-modified pullulan (CHP),which is a type of delivery system, and administered as a vaccine to amouse. With the long chain peptide vaccine adopting Y₆ or T₆ as theinterepitope sequence, specific CD8⁺ T cells corresponding to all threetypes of epitopes were clearly induced. On the other hand, with thevaccine using G₆ or P₆ as the interepitope sequence, the induction ofspecific CD8⁺ T cells corresponding to all three types of epitopes wasclearly weak. From this, it was revealed that the interepitope sequencestrongly influences T cell induction by the preceding and subsequentepitopes and that consecutive tyrosines or threonines is preferable asthe interepitope sequence.

The influences of differences in interepitope sequence of long chainpeptide antigens on antitumor effects of vaccines were examined using amouse tumor implant model (FIG. 2). The long chain peptide antigens MEN,all containing three types of mouse CD8⁺ T cell epitope sequences (MAp265, NY p81, and mERK2 9m), were synthesized, and the sequence betweenthe respective epitopes was set to one of six consecutive tyrosines (Y₆)glycines (G₆), or prolines (P₆). Each long chain peptide antigen wascomplexed with CHP and administered in a single dose as a vaccine to amouse. As a control, a short chain peptide vaccine, constituted of justthe mERK2 9m peptide, was administered. On the day after administration,the mouse fibrosarcoma cell line CMS5a, presenting the CD8⁺ T cellepitope mERK2 9m derived from the mERK2 antigen, was implantedsubcutaneously and its growth was recorded over time. With the vaccineusing the long chain peptide antigen MEN (Y₆), the growth of the tumorwas suppressed significantly (p<0.05). In comparison, the vaccine usingMEN (G₆) or MEN (P₆) or the mERK2 9m short chain peptide vaccine,significant suppression of tumor growth was not observed. It is believedthat the differences in specific CD8⁺ killer T cell induction resultingfrom the differences interepitope sequence seen in FIG. 1 significantlyinfluenced the therapeutic effect due to the vaccines. It was alsorevealed that when an optimal interepitope sequence is adopted, a longchain peptide vaccine exhibits a therapeutic effect that outperforms ashort chain peptide vaccine (in the present case, the vaccine having themERK2 9m peptide as the antigen).

The usefulness of the consecutive tyrosine sequence as an interepitopesequence when the order of epitopes on the long chain peptide antigendiffers from that in the case of FIG. 1, that is, when the epitopesequences preceding and subsequent the interepitope sequence differ wasexamined. The long chain peptide antigens NME, all containing threetypes of mouse CD8⁺ T cell epitope sequences (MA p265, NY p81, and mERK29m), were synthesized. The NME differ from the MEN in FIG. 1 in theorder of the three types of epitopes. The sequence between therespective epitopes was set to one of six consecutive tyrosines (Y₆),glycines (G₆), or prolines (P₆). When vaccines containing the respectivelong chain peptide antigens were administered to mice, specific CD8⁺ Tcells corresponding to all three types of epitopes were clearly inducedwith the vaccine using Y₆ as the interepitope sequence in the samemanner as in FIG. 1 (FIG. 3). On the other hand, with the vaccine usingG₆ or P₆, specific CD8⁺ T cell induction was not observed for one or twotypes of epitopes among the three types of epitopes. To further examinethe influence of the epitope sequences preceding and subsequent theinterepitope sequence, the long chain peptide antigens MEN, ENM, andNME, which are changed in the order of the three types of epitopesequences (MA p265, NY p81, and mERK2 9m) but with which theinterepitope sequence is fixed at six consecutive tyrosines (Y₆), wereprepared. When vaccines containing the respective long chain peptideantigens were administered to mice, specific CD8⁺ T cells correspondingto all three types of epitopes were clearly induced with all of the longchain peptide antigens (FIG. 4). It was thus revealed that with a longchain peptide antigen containing the interepitope sequence constitutedof consecutive tyrosines, specific CD8⁺ T cells for all epitopes can beinduced regardless of the sequences preceding and subsequent theinterepitope sequence.

In many cases with a long chain peptide vaccine, a native amino acidsequence of the protein that is the target antigen is used as it is asthe sequence of the long chain peptide antigen. On the other hand, testresults up to now have revealed that, depending on the sequence betweenepitopes, the preceding and subsequent epitopes do not functionappropriately (FIGS. 1 to 3). It was thus considered that even if theinterepitope sequence is a native amino acid sequence, it may have anunfavorable influence on the functions of the preceding and subsequentepitopes. Thus, the long chain peptide antigen ESO1 LP (native type) anda long chain peptide antigen ESO1 LP (Y₆), both containing a mouse CD8⁺T cell epitope sequence (NY p81 or NY p82) and a mouse CD4⁺ T cellepitope sequence (NY p91) that are derived from human NY-ESO-1 antigen,were synthesized. As the sequence between epitopes, the native aminoacid sequence of NY-ESO-1 was retained with ESO1 LP (native type) andthe sequence of six consecutive tyrosines (Y₆) was used with ESO1 LP(Y₆). When vaccines containing the respective long chain peptideantigens were administered to mice, whereas the induction of NY p81specific CD8⁺ T cells was hardly observed with the vaccine having ESO1LP (native type) as the antigen, the induction was significant with thevaccine having ESO1 LP (Y₆) as the antigen (FIG. 5). Although ESO1 LP(native type) and ESO1 LP (Y₆) differ in containing NY p81 (RGPESRLL(Sequence No. 29)) and NY p82 (GPESRLL (Sequence No. 35)), respectively,as the CD8⁺ T cell epitope, it has been confirmed that NY p82 is poorerin immunogenicity than NY p81 (Non-Patent Document 8) and the excellenceof ESO1 LP (Y₆) over ESO1 LP (native type) is not due to thisdifference. Also, the induction of NY p91 specific CD4⁺ T cells wasclearly observed with both ESO1 LP (native type) and ESO1 LP (Y₆). Fromthe above, it has been revealed that there are cases where aninterepitope sequence derived from a native amino acid sequence does notfunction and that this problem can be resolved by selecting aconsecutive tyrosine sequence as the interepitope sequence.

Deeming that a sequence of consecutive tyrosines is useful as aninterepitope sequence, the optimal number thereof was examined. The longchain peptide antigens MEN, all containing three types of mouse CD8⁺ Tcell epitope sequences (MA p265, NY p81, and mERK2 9m), weresynthesized. The sequence between the respective epitopes was set to oneto six (FIG. 6) or five to ten (FIG. 7) consecutive tyrosines. Vaccinescontaining the respective long chain peptide antigens were administeredto mice in the same manner as in FIG. 1 and the frequencies of theinduced CD8⁺ T cells were measured. In the case of comparing one to sixtyrosines (FIG. 6), in regard to MA p265, which is the first epitope,and NY p81, which is the third epitope, the induction of specific CD8⁺ Tcells was highest when the interepitope sequence was six tyrosines (Y₆)and there was a tendency for CD8⁺ T cell induction to weaken withdecrease in the number of tyrosines. In regard to mERK2 9m, which is thesecond epitope, the induction of specific CD8⁺ T cells was highest whenthe interepitope sequence was four tyrosines (Y₄) and there was atendency for CD8⁺ T cell induction to weaken with decrease in the numberof tyrosines. With this mERK2 9m, when the interepitope sequence was sixtyrosines (Y₆), although lower than that in the case of four tyrosines(Y₄), induction of specific CD8⁺ T cells was observed to some degree. Inview of the above results for the three types of epitopes, it wasconsidered that six is best as the number of tyrosines of theinterepitope sequence, four comes next and is satisfactory, and three orless is not favorable. In the case of comparing five to ten tyrosines(FIG. 7), in regard to MA p265, which is the first epitope, a clearinfluence of the number of tyrosines of the interepitope sequence on theinduction of specific CD8⁺ T cells was not observed. In regard to mERK29m, which is the second epitope, the induction of specific CD8⁺ T cellswas highest when the interepitope sequence was six tyrosines (Y₆) andthere was a tendency for CD8⁺ T cell induction to weaken when the numberof tyrosines was other than six. In regard to NY p81, which is the thirdepitope, a clear influence of the number of tyrosines on the inductionof specific CD8⁺ T cells was not observed with the exception of the casewhere the number of tyrosines of the interepitope sequence was eight(Y₈). In view of the results for the three types of epitopes, it wasconsidered that six is best as the number of tyrosines of theinterepitope sequence and results do no change much even if the numberincreases further. From the above, it was revealed that as the number ofconsecutive tyrosines as the interepitope sequence, four to eight ispreferable, four to six is more preferable, and six is especiallypreferable.

Deeming that a difference in interepitope sequence influences specific Tcell induction by the preceding and subsequent epitopes, a mechanismtherefor is believed to be based on whether or not the interepitopesequence is appropriately cleaved by proteasomes, etc., within anantigen-presenting cell (Non-Patent Document 7). In order to exploreother mechanisms, long chain peptide antigens MEN, with the interepitopesequence being set to one of six consecutive tyrosines (Y₆), glycines(G₆), or prolines (P₆), were synthesized. Each long chain peptideantigen, labeled with the fluorescent dye FAM, was complexed with CHPand administered in vitro to mouse spleen cells including mousedendritic cells and macrophages. Upon measuring the fluorescence uptakesinto the dendritic cells and macrophage, the unexpected finding that theuptake into cells differs according to differences in the interepitopesequence was obtained (FIG. 8). That is, the long chain peptide antigenadopting Y₆ as the interepitope sequence was significantly higher inuptake into cells for both dendritic cells and macrophages in comparisonto cases of G₆ and P₆. A similar finding was also obtained in the testusing individual mice. The same FAM-labeled long chain peptide antigensas those in FIG. 8 were complexed with CHP and administeredsubcutaneously to mice. The fluorescence uptakes into the dendriticcells and mouse macrophages present in the regional lymph node of theadministration site were measured. With the macrophages, the uptake ofthe long chain peptide antigen adopting the Y₆ interepitope sequence wasclearly observed. In contrast, the uptakes of long chain peptideantigens adopting G₆ and P₆ were hardly observed. With the dendriticcells, uptake was not observed for any of the long chain peptideantigens. Together with the results in FIG. 8, it was revealed that along chain peptide antigen with a sequence of consecutive tyrosines asthe interepitope sequence is improved in uptake into antigen-presentingcells, especially macrophages. This phenomenon is likely to be mechanismfor the highly specific T cell induction ability and excellent cancertreatment effect of a vaccine using a long chain peptide antigen with asequence of consecutive tyrosines as the interepitope sequence.

That with vaccines having a long chain peptide, containing a pluralityof T cell epitopes, as an antigen, differences in interepitope sequenceinfluence the success or failure of specific T cell induction by therespective epitopes was examined in in vitro antigen presentationreactions using human immunocytes (FIG. 10). Long chain peptide antigensNMW, all containing the three types of human CD8⁺ T cell recognitionepitope sequences, NY p157, MA4 p143, and WT1 p235, derived from thehuman tumor antigens, NY-ESO-1, MAGE-A4, and WT1, were synthesized. Thesequence between the three types of epitopes was set to that in whichsix of one of alanine (A), glutamic acid (E), glycine (G), histidine(H), asparagine (N), proline (P), glutamine (Q), serine (S), or tyrosine(Y) are made consecutive. A long chain peptide containing aninterepitope sequence constituted of an amino acid besides the above wasdifficult to synthesize or difficult to complex with CHP. Immortalizedhuman B cell lines (LCL) administered with vaccines prepared bycomplexing the respective long chain peptide antigens with CHP were usedas antigen-presenting cells to evaluate the antigen presenting activitywith respect to NY p157 specific CD8⁺ T cell clone 1G4 cells by theIFN-γ ELISPOT method. As with the examination results with mice, whereasthe activation of 1G4 cells was clearly confirmed with the long chainpeptide vaccine adopting the Y₆ interepitope sequence, the activation of1G4 cells was not clearly confirmed with the vaccine adopting the G₆ orthe P₆ interepitope sequence. Also, as with the vaccine using Y₆, theactivation of 1G4 cells was clearly observed with vaccines using A₆, N₆,Q₆, and S₆ as the interepitope sequences and it was thus revealed thatthese interepitope sequences are also useful.

That with vaccines using mRNA encoding a long chain peptide antigen thatcontains a plurality of T cell epitopes, differences in interepitopesequence influence the success or failure of specific T cell inductionby the respective epitopes was examined in in vitro antigen presentationreactions using human immunocytes (FIG. 11). mRNAs that code long chainpeptide antigens NMW, all containing the three types of human CD8⁺ Tcell recognition epitope sequences, NY p157, MA4 p143, and WT1 p235,derived from the human tumor antigens, NY-ESO-1, MAGE-A4, and WT1, weresynthesized. The sequence between the three types of epitopes was set tothat in which six of one of glycine (G), proline (P), threonine (T), ortyrosine (Y) are made consecutive. LCL with the respective mRNAsintroduced therein were used as antigen-presenting cells to evaluate theantigen presenting activity with respect to NY p157 specific CD8⁺ T cellclone 1G4 cells or MA4 p143 specific CD8⁺ T cell clone RNT007#45 cellsby the IFN-γ ELISPOT method. Activations of 1G4 cells and RNT007#45 wereclearly confirmed with the RNA vaccine encoding the long chain peptideadopting Y₆ as the interepitope sequence. From this, it has beenrevealed that the interepitope sequence of the present invention isuseful not only in peptide vaccines but also in RNA vaccines.

The usefulness of consecutive tyrosines or threonines as an interepitopesequence is not limited to the long chain peptide vaccines describedabove and may also be applied to DNA vaccines, mRNA vaccines, ordendritic cell vaccines.

A DNA vaccine may be prepared by using artificial gene synthesistechniques to synthesize a cDNA, encoding a long chain peptide antigenhaving a single methionine at the N-terminus and having a plurality of Tcell epitopes linked by consecutive tyrosine sequences or consecutivethreonine sequences, and inserting it into a gene expression plasmidvector for mammals. The cDNA of the long chain peptide antigen issynthesized to be in the range of 66 to several kbp according to thenumber of T cell epitopes to be included. As the plasmid, that whichcontains pcDNA3, pVAX, or other promoter (CMV promoter, etc.) thatoperates in mammalian cells, polyA (derived from bovine growth hormone,etc.) for mRNA stabilization, and a drug resistance gene (such as thatfor kanamycin) may be used. The plasmid may carry a plurality of longchain peptide antigen cDNAs and the respective antigen cDNAs can beco-expressed by linking with an IRES sequence, etc. Similarly, theplasmid may carry, at the same time, accessory genes for enhancing tumorimmune response, for example, cytokines such as IFN-γ and IL-12,immunostimulatory molecules, such as GITR ligand-Fc, immunosuppressioninhibitors, such as PD-L1-Fc. Also, a plurality of plasmid DNAs thatdiffer in the numbers and types of antigen cDNAs and accessoriesmolecules carried may be administered at the same time.

The DNA vaccine that is obtained is repeatedly administeredsubcutaneously, intradermally, intravenously, intramuscularly,intralymphnodally, epicutaneously, or intratumorally to the living bodyof an animal, such as a mouse (BALB/c mouse or C57BL/6 mouse, etc.), ora human, etc., at a dose of 1 μg to 1 mg per individual and an intervalof one to four weeks using an administration technique such as a genegun, needle-free injector, electroporation method, DNA tattooing,delivery system (cationic liposome, polyethylene imine, etc.),hydrodynamic method, transdermal administration method. One to two weeksafter administration, the specific T cells induced by the T cellepitopes contained in the long chain peptide antigens that aretranscribed and translated from the cDNA on the DNA vaccine may bedetected by an immunological technique such as an intracellular cytokinestaining method, ELISPOT method, MHC tetramer staining method. In testsusing mice, CMS5a fibrosarcoma, CT26 colorectal cancer, 4T1 breastcancer (hereabove in the case of BALB/c mouse), B16 melanoma, or LLClung cancer (hereabove in the case of C57BL/6 mouse) incorporating awild type or model antigen gene may be implanted subcutaneously toobserve the inhibitory effect of the DNA vaccine against growth andmetastasis of the tumor. Tumor growth may be measured by measuring thesize of the tumor or, if tumor cells incorporating a monitor gene suchas a luciferase gene, are used, by an in vivo imaging technique, such asIVIS (PerkinElmer Inc.), etc. To evaluate metastasis, tumor nodules,which, upon intravenous or subcutaneous administration of tumor, occurin the lungs, etc., that are the metastasis destinations, may bevisually counted after dissection or be evaluated by an in vivo imagingtechnique.

With a DNA vaccine, a biological vector using a virus or microorganismmay be used instead of a plasmid vector. As a viral vector, a retroviralvector, lentiviral vector, adenoviral vector, adeno-associated virusvector, vaccinia virus vector, fowlpox virus vector, alphavirus vector,or Sendai virus vector, etc., may be used. As a microorganism vector,yeast, listeria, salmonella, E. coli, or lactobacillus, etc., may beused. A DNA vaccine using such a biological vector is administeredintravenously, subcutaneously, intradermally, intramuscularly,intralymphnodally, supramucosally, or intratumorally to a test animal,such as a mouse, or a human. The arrangement and evaluation methods(immunogenicity and therapeutic effects) of the genes carried on thebiological vector are the same as in the example of the plasmid vectordescribed above.

An mRNA vaccine encoding consecutive tyrosines or threonines as theinterepitope sequence may be implemented in the same manner as a DNAvaccine. Artificial gene synthesis techniques are used to synthesize acDNA, encoding a long chain peptide antigen having a single methionineat the N-terminus and having a plurality of T cell epitopes linked byconsecutive tyrosine sequences or consecutive threonine sequences, andinserting it into a template plasmid DNA for in vitro transfer. The cDNAis prepared to be in the range of 66 to several kbp according to thenumber of T cell epitopes to be included. As the plasmid DNA, that whichcontains a promoter (T7 promoter, T3 promoter, SP6 promoter, etc.)recognized by a phage RNA polymerase, polyA, and a drug resistance gene(such as that for kanamycin), that is for example, pGEM or pcDNA3, etc.,may be used. Using this plasmid DNA as a template, an mRNA issynthesized using a commercially available in vitro transfer kit(MEGAscript, made by Life Technologies, Inc., or RiboMax Large Scale RNAProduction Systems, made by Promega Corporation, etc.). polyA is addedto the mRNA as necessary using a polyA tailing kit (Life Technologies,Inc.), etc. The mRNA obtained is administered subcutaneously,intradermally, intramuscularly, intralymphnodally, or intratumorally asit is or upon stabilizing with a protamine or liposome, etc., to a testanimal, such as a mouse, or a human. The mRNA vaccine may contain aplurality of mRNAs. For example, a plurality of mRNAs that code longchain peptide antigens may be administered upon mixing. An mRNA encodingaccessory molecules for enhancing tumor immune response, for example,cytokines such as IFN-γ and IL-12, immunostimulatory molecules, such asCD40 ligand and GITR ligand-Fc, immunosuppression inhibitors, such asPD-L1-Fc, may be administered at the same time as the mRNA vaccine. Theadministration conditions and evaluation methods (immunogenicity andtherapeutic effects) of the mRNA vaccine are the same as in the exampleof the DNA vaccine described above.

Dendritic cells to be used in a dendritic cell vaccine may be induced todifferentiate in vitro from peripheral blood mononuclear cells in thecase of humans and bone marrow cells in the case of mice by aconventional method using GM-CSF and IL-4. A long chain peptide antigendescribed above or an mRNA encoding a long chain peptide antigendescribed above is added to the cells to prepare a vaccine. If a longchain peptide antigen is used, the efficiency of uptake and expressioncan be increased by using CHP as a delivery system (FIG. 8). If an mRNAencoding a long chain peptide antigen is used, the efficiency of uptakeand expression in dendritic cells can be increased by electroporationmethod. In this process, an mRNA encoding an accessory molecule forenhancing tumor immune response may be added at the same time asdescribed above. The dendritic cells after addition of antigen may beused upon being stimulated and matured by TNFα, IL-1β, IL-6, Flt3ligand, PGE₂, CpG oligo DNA, poly IC RNA, etc. The dendritic cellvaccine obtained is administered subcutaneously, intradermally,intralymphnodally, intratumorally, or intravenously to a test animal,such as a mouse, or a human at a dose of 10⁶ to 10⁸ cells. Theevaluation methods (immunogenicity and therapeutic effects) are the sameas in the example of the DNA vaccine described above.

Along chain peptide vaccine, DNA vaccine, mRNA vaccine, or dendriticcell vaccine adopting consecutive tyrosines or threonines as theinterepitope sequence may be applied to diseases other than cancer, forexample, to infectious diseases. As pathogens of infections, pathogenicviruses, such as hepatitis virus, human papilloma virus, adult T-cellleukemia virus, human immunodeficiency virus, herpes virus, influenzavirus, Coxsackie virus, rotavirus, RS virus, varicella zoster virus,measles virus, polio virus, norovirus, pathogenic obligate intracellularparasitic microorganisms, such as rickettsia, chlamydia, phytoplasma,Coxiella, Toxoplasma, Leishmania, protozoa, such as Plasmodium,Cryptosporidium, can be cited.

For example, for a vaccine against the hepatitis C virus, a long chainpeptide antigen may be designed with which a plurality of T cellepitopes, identified in hepatitis C virus-derived proteins, such as thecore protein, NS4, and NS3, are linked with an interepitope sequenceconstituted of consecutive tyrosines or threonines. Administrationconditions of the vaccine containing the long chain peptide antigen andtherapeutic effects on hepatitis C virus infection may be examined usinga model system, such as an immunodeficient mouse transplanted with humanliver tissue. Similarly, for a vaccine against human herpesvirus, a longchain peptide antigen may be designed using T cell epitopes contained inthe human herpesvirus-derived proteins E6 and E7, and administrationconditions and therapeutic effects may be examined with a mouse modeltransplanted with a tumor that expresses E6 or E7. For a vaccine againsta pathogenic microorganism, for example, for a vaccine against malaria,a long chain peptide antigen is designed with which a group of T cellepitopes, contained in merozoite surface protein 3 (MSP3) and glutamaterich protein (GLURP), which are expressed on the surface of the maturebody of Plasmodium, and liver-specific protein 2 (LISP2), which isexpressed in the intracanal air, are linked with a sequence ofconsecutive tyrosines or a sequence of consecutive threonines. A mouseadministered with a vaccine containing the long chain peptide antigen,is intravenously administered with 10,000 Plasmodium sporozoites and aperipheral blood smear is prepared 4 to 14 days later. Administrationconditions and therapeutic effects of the vaccine may be examined bystaining with Giemsa and thereafter observing the parasitemia under amicroscope.

The above results show on one hand that differences in interepitopesequence have a large influence on specific T cell induction by aplurality of epitopes contained in a long chain peptide antigen and thata cancer treatment vaccine using an inappropriate interepitope sequencesis poor in inducing the intended T cells and in cancer treatment effect,and show on the other hand that by using consecutive tyrosines orthreonines as the interepitope sequence, specific T cell induction bythe plurality of epitopes contained in the long chain peptide antigencan be achieved reliably and a cancer treatment vaccine that exhibitshigh treatment effects can be realized. In the process, it has beenrevealed that the effects are exhibited regardless of the epitopesequences preceding and subsequent the interepitope sequence and it wasalso possible to define the optimal length of the interepitope sequence.

According to the present embodiments, it was possible to provide cancertreatment vaccines of extremely high cancer treatment effects.

1. A vaccine including a long chain peptide antigen having a pluralityof epitopes in which each interepitope sequence is one selected from agroup consisting of two to ten consecutive tyrosines, two to tenconsecutive threonines, two to ten consecutive alanines, two to tenconsecutive histidines, two to ten consecutive glutamines and two to tenconsecutive asparagines.
 2. The vaccine as claimed in claim 1, which isone selected from a group consisting of anticancer vaccines,antibacterial vaccines and antiviral vaccines.
 3. The vaccine as claimedin claim 2, which is one selected from a group consisting of peptidevaccines, DNA vaccines, mRNA vaccines and dendritic cell vaccines. 4.The vaccine as claimed in claim 3, wherein the interepitope sequence isone selected from a group consisting of four to eight consecutivetyrosines, four to eight consecutive glutamines and four to eightconsecutive asparagines.
 5. The vaccine as claimed in claim 3, whereinthe interepitope sequence is one selected from a group consisting of sixconsecutive tyrosines, six consecutive glutamines and six consecutiveasparagines.
 6. The vaccine as claimed in claim 4, which is ananticancer vaccine and a peptide vaccine.
 7. The vaccine as claimed inclaim 4, which contains a plurality of long chain peptide antigens.
 8. Acombination vaccine which contains the vaccine as claimed in claim 7 anda hydrophobized polysaccharide as a delivery system.
 9. The vaccine asclaimed in claim 8, wherein the hydrophobized polysaccharide ischolesterol-modified pullulan (CHP).
 10. A method of treating a patientin need thereof, comprising administering to the patient the vaccine ofclaim
 9. 11. A method of treating a patient in need thereof, comprisingadministering to the patient the vaccine of claim
 1. 12. The vaccine asclaimed in claim 1, which is one selected from a group consisting ofpeptide vaccines, DNA vaccines, mRNA vaccines and dendritic cellvaccines.
 13. The vaccine as claimed in claim 1, wherein theinterepitope sequence is one selected from a group consisting of four toeight consecutive tyrosines, four to eight consecutive glutamines andfour to eight consecutive asparagines.
 14. The vaccine as claimed inclaim 1, wherein the interepitope sequence is one selected from a groupconsisting of six consecutive tyrosines, six consecutive glutamines andsix consecutive asparagines.
 15. The vaccine as claimed in claim 1,which is an anticancer vaccine and a peptide vaccine.
 16. The vaccine asclaimed in claim 1, which contains a plurality of long chain peptideantigens.
 17. A combination vaccine which contains the vaccine asclaimed in claim 1 and a hydrophobized polysaccharide as a deliverysystem.
 18. The vaccine as claimed in claim 17, wherein thehydrophobized polysaccharide is cholesterol-modified pullulan (CHP).